Vegf antagonist formulations

ABSTRACT

Formulations of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist are provided including a pre-lyophilized formulation, a reconstituted lyophilized formulation, and a stable liquid formulation. Preferably, the fusion protein has the sequence of SEQ ID NO:4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/387,256, filed 22 Mar. 2006, which claims the benefit of priorityunder 35 USC §119(e) of U.S. Provisional Application No. 60/665,125,filed 25 Mar. 2005, which applications are herein specificallyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to pharmaceutical formulationscomprising agents capable of inhibiting vascular endothelial growthfactor (VEGF), and to methods for making and using such formulations.The invention includes pharmaceutical formulations having increasedstability.

2. Statement of Related Art

Vascular endothelial growth factor (VEGF) expression is nearlyubiquitous in human cancer, consistent with its role as a key mediatorof tumor neoangiogenesis. Blockade of VEGF function, by binding to themolecule or its VEGFR-2 receptor, inhibits growth of implanted tumorcells in multiple different xenograft models (see, for example, Gerberet al. (2000) Cancer Res. 60:6253-6258). A soluble VEGF-specific fusionprotein antagonist, termed a “VEGF trap” has been described (Kim et al.(2002) Proc. Natl. Acad. Sci. USA 99:11399-404; Holash et al. (2002)Proc. Natl. Acad. Sci. USA 99:11393-8), which references arespecifically incorporated by reference in their entirety.

Lyophilization (freeze drying under controlled conditions) is commonlyused for long term storage of proteins. The lyophilized protein issubstantially resistant to degradation, aggregation, oxidation, andother degenerative processes while in the freeze-dried state (see, forexample, U.S. Pat. No. 6,436,897).

BRIEF SUMMARY OF THE INVENTION

Stable formulations of a VEGF-specific fusion protein antagonist areherein provided. The pharmaceutically acceptable formulations of theinvention comprise the VEGF “trap” antagonist with a pharmaceuticallyacceptable carrier. In specific embodiments, liquid and freeze-dried, orlyophilized formulations are provided.

In a first aspect, the invention features a stable liquid formulation ofa VEGF-specific fusion protein antagonist, comprising a fusion proteincomprising a receptor component consisting essentially of animmunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain3 of a second VEGF receptor, and a multimerizing component, one or morebuffers, and one or more thermal stabilizers. In a specific embodimentof the VEGF-specific fusion protein antagonist, the first VEGF receptoris Flt1 and the second VEGF receptor is Flk1 or Flt4. In a more specificembodiment the fusion protein has the amino acid sequence of SEQ ID NO:2or SEQ ID NO:4. In one embodiment, the buffer is a phosphate bufferand/or citrate. More preferably, the buffers are phosphate and citrate.In one embodiment, the thermal stabilizers are NaCl and/or sucrose. Morepreferably, the thermal stabilizers are both NaCl and sucrose.

In a specific embodiment, the stable liquid formulation of aVEGF-specific fusion protein antagonist comprises 1-10 mM phosphatebuffer, 1-10 mM citrate, 25-150 mM NaCl, 5-30% sucrose, 10-50 mg/ml ofthe fusion protein, at a pH of about 6-6.5. In a more specificembodiment, the stable liquid formulation comprises 5 mM phosphatebuffer, 5 mM citrate buffer, 100 mM NaCl, 20% sucrose, 25 mg/ml of thefusion protein, at a pH of about 6.0. Additionally, polysorbate may bepresent, for example 0.05-0.15% polysorbate 20. The stable liquidformulation of the VEGF-specific fusion protein antagonist of theinvention exhibits little or no precipitation after storage of a 25mg/ml VEGF formulation for about 6 months at −80° C. and little or noprecipitation after storage for 6 months at 5° C.

In a second aspect, the invention features a high concentration stableliquid formulation of a VEGF antagonist comprising 1-50 mM histidine,25-150 mM NaCl, 5-30% sucrose, 50-100 mg/ml of the fusion protein, at apH of about 6-6.5, and either 0.1-0.5% polysorbate or 1-5% PEG. In amore specific embodiment, the high concentration stable liquidformulation comprises 10 mM histidine, 50 mM NaCl, 5-20% sucrose, 50-100mg/ml of the fusion protein, at a pH of about 6.0-6.5, with either 0.1%polysorbate (e.g., polysorbate 20) or 3% PEG (e.g., PEG 3350). The highconcentration stable liquid formulation of the VEGF-specific fusionprotein antagonist of the invention exhibits less than about 3%degradation after 15 months of storage at 5° C. (75 or 100 mg/ml VEGFtrap protein) or less than about 1.5% degradation after 24 months (50mg/ml).

In a third aspect, the invention features a pre-lyophilized formulationof a vascular endothelial growth factor (VEGF)-specific fusion proteinantagonist, comprising a (i) fusion protein comprising a receptorcomponent consisting essentially of an immunoglobulin-like (Ig) domain 2of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, anda multimerizing component, (ii) a buffer, (iii) an organic co-solvent orbulking agent, and (iv) one or more lyoprotectants. In variousembodiments, the buffer is histidine, the organic co-solvent or bulkingagent is PEG, and the lyoprotectant(s) is at least one of glycine andsucrose. In one embodiment, the pre-lyophilized formulation of theinvention does not contain a preservative.

In one embodiment of the pre-lyophilized formulation of the invention,the formulation comprises 5-50 mM histidine, 0.1-3.0% PEG, 0.25-3.0%glycine, 0.5-6.0% sucrose, and 5-75 mg/ml of the fusion protein, at a pHof about 6.0-6.5. In any embodiment, the pre-lyophilized formulation mayfurther comprise up to 0.05 mM citrate and/or 0.003-0.005% polysorbate.The polysorbate present may be, for example, polysorbate 20.

In a more specific embodiment, the pre-lyophilized formulation comprisesabout 10 mM histidine, about 1.5% PEG 3350, about 0.75% glycine, about2.5% sucrose, and about 12.5 to 75 mg/ml VEGF-specific fusion protein,at a pH of about 6.25. In specific embodiments, the fusion proteincomprises the protein sequence of SEQ ID NO:4, present as a multimer,e.g., a dimer. In separate embodiments, the reconstituted formulation is2 times the concentration of the pre-lyophilized formulation, e.g., a 20mg fusion protein/ml pre-lyophilized formulation is reconstituted to afinal formulation of 60 mg fusion protein/ml. Generally, the lyophilizedformulation is reconstituted with sterile water suitable for injection.In one embodiment, the reconstitution liquid may be bacteriostaticwater.

In a preferred embodiment, the pre-lyophilized formulation consistsessentially of about 10 mM histidine, about 1.5% PEG 3350, about 0.75%glycine, about 2.5% sucrose, and about 50 mg/ml of the fusion proteinhaving the sequence of SEQ ID NO:4 as a dimer, at a pH of about 6.25.Citrate (less than or equal to about 0.02 mM) and/or polysorbate (lessthan or equal to about 0.0005%) may be present. Optionally, thepre-lyophilized formulation does not contain a preservative, a phosphatebuffer, and/or more than trace amounts of NaCl. In one embodiment, thepre-lyophilized formulation consists of about 10 mM histidine, about1.5% PEG 3350, about 0.75% glycine, about 2.5% sucrose, and about 50mg/ml of the VEGF trap protein (SEQ ID NO:4), pH 6.3, and uponreconstitution contains 20 mM histidine, 3% PEG, 1.5% glycine, about 5%sucrose, and about 100 mg/ml VEGF trap protein.

In a fourth aspect, the invention features a method of producing alyophilized formulation of a VEGF-specific fusion protein antagonist,comprising subjecting the pre-lyophilized formulation of the inventionto lyophilization to generate a lyophilized formulation. The lyophilizedformulation may be lyophilized by any method known in the art forlyophilizing a liquid.

In a fifth related aspect, the invention features a method of producinga reconstituted lyophilized formulation of a VEGF-specific fusionprotein antagonist, comprising reconstituting the lyophilizedformulation of the invention to a reconstituted formulation. In oneembodiment, the reconstituted formulation is twice the concentration ofthe pre-lyophilized formulation, e.g., the method of the inventioncomprises: (a) producing a pre-lyophilized formulation of aVEGF-specific fusion protein antagonist, (b) subjecting thepre-lyophilized formulation of step (a) to lyophilization; and (c)reconstituting the lyophilized formulation of step (b).

In specific embodiments of the method of producing a reconstitutedlyophilized formulation, a pre-lyophilized solution is present in a vialas a 25 mg VEGF-specific fusion protein antagonist per ml solution ofpre-lyophilized formulation, which is lyophilized and reconstituted toan 50 mg/ml solution. In another embodiment, a 30 mg/ml pre-lyophilizedsolution is lyophilized and reconstituted to a 60 mg/ml solution. Inanother embodiment, a 40 mg/ml pre-lyophilized solution is lyophilizedand reconstituted to a 80 mg/ml solution. In another embodiment, a 12.5mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 25mg/ml solution. In another embodiment, a 50 mg/ml pre-lyophilizedsolution is lyophilized and reconstituted to a 100 mg/ml solution. Inanother embodiment, a 75 mg/ml pre-lyophilized solution is lyophilizedand reconstituted to a 150 mg/ml solution. Preferably, the reconstitutedlyophilized formulation does not contain a preservative.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is not limited to particular methods, andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting unless indicated, since the scope of the presentinvention will be limited only by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “a method”include one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless stated otherwise, all technical and scientific terms and phrasesused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference.

General Description

Safe handling and administration of formulations comprising proteinsrepresent significant challenges to pharmaceutical formulators. Proteinspossess unique chemical and physical properties that present stabilityproblems: a variety of degradation pathways exist for proteins,implicating both chemical and physical instability. Chemical instabilityincludes deamination, aggregation, clipping of the peptide backbone, andoxidation of methionine residues. Physical instability encompasses manyphenomena, including, for example, aggregation.

Chemical and physical stability can be promoted by removing water fromthe protein. Lyophilization (freeze-drying under controlled conditions)is commonly used for long-term storage of proteins. The lyophilizedprotein is substantially resistant to degradation, aggregation,oxidation, and other degenerative processes while in the freeze-driedstate. The lyophilized protein is normally reconstituted with wateroptionally containing a bacteriostatic preservative (e.g., benzylalcohol) prior to administration.

DEFINITIONS

The term “carrier” includes a diluent, adjuvant, excipient, or vehiclewith which a composition is administered. Carriers can include sterileliquids, such as, for example, water and oils, including oils ofpetroleum, animal, vegetable or synthetic origin, such as, for example,peanut oil, soybean oil, mineral oil, sesame oil and the like.

The term “excipient” includes a non-therapeutic agent added to apharmaceutical composition to provide a desired consistency orstabilizing effect. Suitable pharmaceutical excipients include, forexample, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like.

The term “lyophilized” or “freeze-dried” includes a state of a substancethat has been subjected to a drying procedure such as lyophilization,where at least 50% of moisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Generally, acceptablebulking agents known to the art include, for example, carbohydrates,including simple sugars such as dextrose, ribose, fructose and the like,alcohol sugars such as mannitol, inositol and sorbitol, disaccharidesincluding trehalose, sucrose and lactose, naturally occurring polymerssuch as starch, dextrans, chitosan, hyaluronate, proteins (e.g., gelatinand serum albumin), glycogen, and synthetic monomers and polymers. Inthe formulations of the invention, PEG 3350 is an organic co-solventwhich is used to stabilize the fusion protein when agitated, mixed, orhandled, and as a bulking agent to help produce an acceptable bulk.

The term “lyoprotectant” includes a substance that may be added to afreeze-dried or lyophilized formulation to help maintain proteinstructure when freeze-dried or lyophilized.

A “preservative” includes a bacteriostatic, bacteriocidal, fungistaticor fungicidal compound that is generally added to formulations to retardor eliminate growth of bacteria or other contaminating microorganisms inthe formulations. Preservatives include, for example, benzyl alcohol,phenol, benzalkonium chloride, m-cresol, thimerosol, chlorobutanol,methylparaben, propylparaben and the like. Other examples ofpharmaceutically acceptable preservatives can be found in the USP.

VEGF Antagonists

An VEGF antagonist is a compound capable of blocking or inhibiting thebiological action of vascular endothelial growth factor (VEGF), andincludes fusion proteins capable of trapping VEGF. In a preferredembodiment, the VEGF antagonist is the fusion protein of SEQ ID NO:2 or4; more preferably, SEQ ID NO:4. In specific embodiments, the VEGFantagonist is expressed in a mammalian cell line such as a CHO cell andmay be modified post-translationally. In a specific embodiment, thefusion protein comprises amino acids 27-457 of SEQ ID NO:4 and isglycosylated at Asn residues 62, 94, 149, 222 and 308.

The VEGF antagonist of the methods and formulations of the invention canbe prepared by any suitable method known in the art, or that comes to beknown. The VEGF antagonist is preferably substantially free of proteincontaminants at the time it is used to prepare the pharmaceuticallyacceptable formulation. By “substantially free of protein contaminants”is meant, preferably, that at least 90% of the weight of protein of theVEGF-specific fusion protein antagonist preparation used for making aformulation is VEGF fusion protein antagonist protein, more preferablyat least 95%, most preferably at least 99%. The fusion protein ispreferably substantially free of aggregates. “Substantially free ofaggregates” means that at least 90% of the weight of fusion protein isnot present in an aggregate at the time the fusion protein is used toprepare the pharmaceutically effective formulation. The fusion proteinof the methods and formulations of the invention may contain low ortrace amounts of compounds as a results of the purification process, forexample, low or trace amounts of citrate and/or polysorbate. In oneembodiment of the pre-lyophilized formulation of the inventioncontaining about 50 mg of fusion protein/ml, citrate may be present at aconcentration of about 0.02 mM and/or polysorbate may be present at aconcentration of about 0.0005%. If the pre-lyophilized formulation isreconstituted after lyophilization to half of the original volume (e.g.,100 mg/ml of fusion protein), the resulting concentrations may be 0.04mM citrate and/or 0.001% polysorbate.

Lyophilization and Lyophilized Formulations

In one aspect of the invention, a pharmaceutically acceptableformulation comprising a VEGF-specific fusion protein antagonist isprovided, wherein the formulation is a freeze-dried or lyophilizedformulation. Lyophilized formulations can be reconstituted intosolutions, suspensions, emulsions, or any other suitable form foradministration or use. Lyophilized formulations are typically firstprepared as liquids, then frozen and lyophilized. The total liquidvolume before lyophilization can be less, equal to, or more than, thefinal reconstituted volume of the lyophilized formulation. Thelyophilization process is well known to those of ordinary skill in theart, and typically includes sublimation of water from a frozenformulation under controlled conditions.

Lyophilized formulations can be stored at a wide range of temperatures.Lyophilized formulations may be stored below 25° C., for example,refrigerated at 4° C., or at room temperature (e.g., approximately 25°C.). Preferably, lyophilized formulations are stored below about 25° C.,more preferably, at about 4-20° C.; below about 4° C.; below about −20°C.; about −40° C.; about −70° C., or about −80° C.

Lyophilized formulations are typically reconstituted for use by additionof an aqueous solution to dissolve the lyophilized formulation. A widevariety of aqueous solutions can be used to reconstitute a lyophilizedformulation. Preferably, lyophilized formulations are reconstitutedusing water. Lyophilized formulations are preferably reconstituted witha solution consisting essentially of water (e.g., USP WFI, or water forinjection) or bacteriostatic water (e.g., USP WFI with 0.9% benzylalcohol). However, solutions comprising buffers and/or excipients and/orone or more pharmaceutically acceptable carries can also be used.

Freeze-dried or lyophilized formulations are typically prepared fromliquids, that is, from solutions, suspensions, emulsions, and the like.Thus, the liquid that is to undergo freeze-drying or lyophilizationpreferably comprises all components desired in a final reconstitutedliquid formulation. As a result, when reconstituted, the freeze-dried orlyophilized formulation will render a desired liquid formulation uponreconstitution. A preferred liquid formulation used to generate afreeze-dried or lyophilized formulation comprises a VEGF-specific fusionprotein antagonist in a pharmaceutically effective amount, a buffer, astabilizer, and a bulking agent. Freeze-dried or lyophilizedformulations preferably comprise histidine, since histidine, incomparison to phosphate, is more effective at stabilizing the fusionprotein when the fusion protein is lyophilized. Organic co-solvents,such as PEG 3350, are used to stabilize the fusion protein whenagitated, mixed, or handled. A lyoprotectant is preferably used infreeze-dried or lyophilized formulations. Lyoprotectants help tomaintain the secondary structure of proteins when freeze-dried orlyophilized. Two preferred example lyoprotectants are glycine andsucrose, which are preferably used together.

Stable Liquid Formulations

In one aspect, the invention provides a stable pharmaceuticallyacceptable formulation comprising a VEGF-specific fusion proteinantagonist, wherein the formulation is a liquid formulation. Preferably,the liquid formulation comprises a pharmaceutically effective amount ofthe fusion protein. The formulation can also comprise one or morepharmaceutically acceptable carriers, buffers, bulking agents,stabilizers, preservatives, and/or excipients. An example of apharmaceutically acceptable liquid formulation comprises a VEGF-specificfusion protein antagonist in a pharmaceutically effective amount, abuffer, a co-solvent, and one or more stabilizers.

A preferred liquid formulation comprises phosphate buffer, an organicco-solvent, and one or more thermal stabilizers to minimize formation ofaggregates and low molecular weight products when stored, and about 10mg/ml to about 50 mg/ml fusion protein, wherein the formulation is fromabout pH 6.0-6.5. A preferred liquid formulation comprises about 5 mMphosphate buffer, about 5 mM citrate, about 100 mM NaCl, about 25%sucrose, and about 10-50 mg/ml fusion protein, wherein the formulationis at a pH of about 6.0; optionally polysorbate may be present (e.g.,0.1% polysorbate 20). Although either NaCl or sucrose can be used as astabilizer, a combination of NaCl and sucrose has been established tostabilize the fusion protein more effectively than either individualstabilizer alone.

Stability is determined in a number of ways at specified time points,including determination of pH, visual inspection of color andappearance, determination of total protein content by methods known inthe art, e.g., UV spectroscopy, SDS-PAGE, size-exclusion HPLC, bioassaydetermination of activity, isoelectric focusing, and isoaspartatequantification. In one example of a bioassay useful for determining VEGFantagonist activity, a BAF/3 VEGFR1/EPOR cell line is used to determineVEGF165 binding by the VEGF-specific fusion protein antagonist of theinvention.

Formulations, whether liquid or freeze-dried and lyophilized, can bestored in an oxygen-deprived environment. Oxygen-deprived environmentscan be generated by storing the formulations under an inert gas such as,for example, argon, nitrogen, or helium.

EXAMPLES

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly to the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described.

Example 1 Stability of a 50 mg/ml Liquid Formulation of VEGF Trap

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 0.1%polysorbate 20, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH6.25, was stored at 5° C. and samples tested at 3, 6, 9, 12, 18 and 24months. Stability was determined by SE-HPLC. The results, shown in Table1, show that 98.6% and 98.3% of VEGF trap protein remained intact(non-degraded) at 12 and 24 months, respectively. Turbidity was measuredat OD₄₀₅ nm; and percent recovered protein by size exclusion HPLC.

TABLE 1 Stability of 50 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS065) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 99.0 3 Pass0.00 6.2 102 98.8 6 Pass 0.01 6.2 103 98.7 9 Pass 0.01 6.3 102 98.2 12Pass 0.01 6.3 106 98.6 18 Pass 0.00 6.3 103 98.4 24 Pass 0.00 6.2 9398.3

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 3% PEG3350, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at 5° C. and samples tested at 3, 6, 9, 12, 18 and 24 months.Stability results are shown in Table 2.

TABLE 2 Stability of 50 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS065) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 99.0 3 Pass0.00 6.2 100 98.8 6 Pass 0.01 6.3 103 98.5 9 Pass 0.00 6.3 103 98.3 12Pass 0.01 6.3 110 98.3 18 Pass 0.00 6.3 113 98.0 24 Pass 0.01 6.2 9097.8

Example 2 Stability of a 75 mg/ml Liquid Formulation of VEGF Trap

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 0.1%polysorbate 20, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH6.25, was stored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and15 months. Stability results are shown in Table 3.

TABLE 3 Stability of 75 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 97.1 1 Pass0.00 6.2 96 97.0 2.3 Pass 0.00 6.2 98 96.7 3 Pass 0.00 6.2 97 96.1 9Pass −0.01 6.0 101 96.0 12 Pass 0.00 6.3 110 94.5 15 Pass 0.00 6.3 9295.6

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 3% PEG3350, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months.Stability results are shown in Table 4.

TABLE 4 Stability of 75 mg/ml VEGF Trap Protein When Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.2 100 96.8 1 Pass0.00 6.2 99 96.7 2.3 Pass 0.00 6.2 97 96.3 3 Pass 0.00 6.2 89 95.6 9Pass −0.01 6.2 98 95.4 12 Pass −0.01 6.3 112 94.1 15 Pass 0.00 6.3 9894.8

Example 3 Stability of a 100 mg/ml Liquid Formulation of VEGF Trap

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 0.1%polysorbate 20, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH6.25, was stored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and15 months. Stability results are shown in Table 5.

TABLE 5 Stability of 100 mg/ml VEGF Trap Protein Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.3 100 96.7 1 Pass0.00 6.2 92 96.6 2.3 Pass 0.00 6.2 92 96.2 6 Pass 0.00 6.2 99 95.5 9Pass −0.01 6.2 92 95.5 12 Pass −0.01 6.2 110 93.9 15 Pass 0.00 6.3 10894.8

A liquid formulation containing 10 mM phosphate, 50 mM NaCl, 3% PEG3350, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at 5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months.Stability results are shown in Table 6.

TABLE 6 Stability of 100 mg/ml VEGF Trap Protein Stored at 5° C.(VGFT-SS101) % VEGF Trap Visual % VEGF Trap Native Months AppearanceTurbidity pH Recovered Configuration 0 Pass 0.00 6.3 100 96.5 1 Pass0.01 6.2 94 96.2 2.3 Pass 0.01 6.2 93 95.7 6 Pass 0.01 6.2 102 94.6 9Pass 0.00 6.2 95 94.6 12 Pass 0.00 6.3 96 92.8 15 Pass 0.01 6.3 102 93.9

Example 4 Further Embodiments of Stable VEGF Trap Formulations

In one embodiment, the invention provides a stable liquid VEGF-bindingfusion protein (VEGF trap) formulations comprising 5 mM phosphate, 5 mMcitrate, 100 mM NaCl, 0.1% Polysorbate 20, 20% sucrose, 25 mg/ml VEGFtrap protein, pH 6.0. This formulation can either be deliveredsubcutaneously or diluted and delivered by intravenous infusion. Due tothe high osmolality of this formulation, it is diluted 3-fold to achievean iso-osmolar solution for intravenous administration. Stabilitystudies showed less than about 1% degradation was detected after 3 yearsof storage at 2-8° C.

In one embodiment, the invention features a lyophilized formulationwhich is preferably concentrated two-fold from the pre-lyophilized tothe post-lyophilized formulation, e.g., 50 to 100 mg/ml; 75 to 150mg/ml, or 100 to 200 mg/ml VEGF trap protein. In one specificembodiment, the pre-lyophilized formulation comprises 10 mM histidine,1.5% PEG 3350, 0.75% glycine, 2.5% sucrose, 50 mg/ml VEGF trap protein,pH 6.3, and is reconstituted to a formulation comprising 20 mMhistidine, 3% PEG 3350, 1.5% glycine, 5% sucrose, 100 mg/ml VEGF trapprotein, pH 6.3. Stability studied showed no degradation of the VEGFtrap was detected after 6 months of storage at 2-8° C.

In one embodiment of a liquid formulation, the formulation comprises 10mM histidine, 50 mM NaCl, 5-20% sucrose, 50-100 mg/ml VEGF trap, and oneof 0.1% polysorbate 20 or 3% PEG 3350. One advantage of this liquidformulation is that it provides a higher concentration of VEGF trapwithout requiring the manufacture of a lyophilized product. Thus, thisformulation provides ease for subcutaneous delivery, for example, byallowing provision of a liquid pre-filled syringe at a concentrationhigher than that delivered by IV infusion. Also, this formulation couldadvantageously be used to provide lower infusion volumes and shorterinfusion times. The amount of degradation determined by SE-HPLCfollowing incubation at 5° C. for up to 15 or 24 months is summarized inTable 7.

TABLE 7 Stability of Liquid Formulation with 50-100 mg/ml VEGF Trap(VGFT-SS101) Incubation VEGF Trap % Polysorbate (months) (mg/ml) 20 %PEG 3350 % Degradation 24 50 0.1 — 0.7 24 50 — 3 1.3 15 75 0.1 — 1.5 1575 — 3 2.0 15 100 0.1 — 1.9 15 100 — 3 2.6

Example 5 Stability and Activity of Lyophilized and Liquid

The stability of a reconstituted lyophilized formulation was determinedover a 6 month period. The pre-lyophilized formulation contained 10 mMhistidine, 1.5% PEG 3350, 2.5% sucrose, 0.75% glycine and 50 mg/ml VEGFtrap protein. After lyophilization, the reconstituted formulationcontained 20 mM histidine, 3% PEG 3350, 5% sucrose, 1.5% glycine, and100 mg/ml VEGF trap protein (SEQ ID NO:4). The results shown in Table 8.Activity was determined in a cell based bioassay which directly measuresthe ability of the VEGF trap to inhibit the biological effects of humanVEGF on a mouse Baf/3 VEGFR1/EpoR cell line. Therefore, this bioassaydirectly measures the biological activity of the protein. The resultsare expresses as percent relative potency (test sample IC₅₀/referenceVEGF IC₅₀ standard×100). The binding affinity of VEGF to the VEGF trapis measured using a sensitive ELISA that specifically measures free VEGFin equilibrated mixtures containing VEGF and various concentrations ofthe VEGF trap. Results are expressed as percent relative binding (IC₅₀of test sample/IC₅₀ of reference×100). Measured pH ranged between6.3-6.5. All solutions where visually clear. The concentration of VEGFtrap recovered was determined with a UV spectrophotometer as mg/ml atA₂₈₀ nm. The percent VEGF trap recovered in the native configuration(main peak purity) was determined with SE-HPLC.

TABLE 8 Stability of VEGF Trap Lyophilized Formulation Stored at 5° C.(VGT-RS475) % Native Months Bioassay Binding Assay % RecoveredConfiguration 0 120 126 97.9 98.7 1 117 74 97.9 98.6 1 + 24 hr 126 7299.0 98.5  1 + 4 hr 94 81 101.5 98.2 3 101 98 98.1 98.6 3 + 24 hr 65 9498.1 98.2  6 + 4 hr 96.9 98.7 6 + 24 hr 98.8 98.6

A formulation containing about 5 mM phosphate, 5 mM citrate, 100 mMNaCl, 0.1% polysorbate 20, 20% sucrose, and 25 mg/ml VEGF trap proteinwas tested for stability and activity over 36 months when stored at 5°C. The results are shown in Table 9. All samples were clear andcolorless as determined by visual inspection. pH ranged from 6.0-6.1.*Binding assay results for two measurements (1 and 2 months) areexpressed directly and not as a percent of the standard.

TABLE 9 Stability and Activity of Liquid Formulation (VGT-FS405) %Native Protein Content Months Configuration Bioassay Binding Assay mg/ml0 99.7 106 72 25.0 1 99.9 119 4.4 pM* 25.2 2 99.6 102 5.4 pM* 25.1 399.6 97 88 25.1 6 99.6 101 106 25.0 9 99.4 89 126 25.4 12 99.5 85 9525.2 18 99.4 99 81 25.5 24 99.3 75 95 25.6 36 98.8 109 79 25.6

1. A stable liquid formulation of a vascular endothelial growth factor(VEGF)-specific fusion protein antagonist, comprising 10-100 mg/ml of afusion protein having a receptor component consisting of animmunoglobulin-like (Ig) domain 2 of Flt1 and Ig domain 3 of Flk1 orFlt4, and a multimerizing component; 1-10 mM phosphate, at a pH of about6.0-6.5; a thermal stabilizer selected from the group consisting ofabout 20% sucrose, about 50-100 mM NaCl, and a combination thereof; andabout 0.1% polysorbate 20 or about 3% PEG 3350; wherein at least 92% ofthe fusion protein antagonist is present in native conformation asmeasured by SE-HPLC following storage at 5° C. for 12 months.
 2. Thestable liquid formulation of claim 1, further comprising 1-10 mMcitrate.
 3. The stable liquid formulation of claim 2, wherein about 99%of the fusion protein antagonist is undegraded following storage at 5°C. for 24 months, and wherein the fusion protein displays about 75-100%of maximal potency in a bioassay in a mouse Baf/3 VEGFR1/EpoR cell line.4. The stable liquid formulation of claim 1, wherein the fusion proteincomprises the sequence of SEQ ID NO:4.
 5. The stable liquid formulationof claim 3, wherein the fusion protein comprises the sequence of SEQ IDNO:4.
 6. The stable liquid formulation of claim 3, comprising 25-50mg/ml of the fusion protein.
 7. The stable liquid formulation of claim5, comprising 5 mM phosphate buffer, 5 mM citrate buffer, 100 mM NaCl,and 25 mg/ml of the fusion protein of SEQ ID NO:4, at a pH of about 6.0.8. The stable liquid formulation of claim 5, comprising 5 mM phosphatebuffer, 5 mM citrate buffer, 100 mM NaCl, and 50 mg/ml of the fusionprotein, at a pH of about 6.0.
 9. A stable liquid formulation of a VEGFantagonist, comprising: (a) about 10 mM histidine; (b) about 50 mM NaCl;(c) about 5-20% sucrose; (d) about 0.1% polysorbate 20 or about 3% PEG3350; and (e) about 50-100 mg/ml of a VEGF antagonist of SEQ ID NO:4, ata pH of about 6.0-6.5, wherein no more than about 2.6% of the fusionprotein is degraded following storage for up to 15 months at about 5°C., as measured by size exclusion chromatography.
 10. The stable liquidformulation of claim 9, comprising about 0.1% polysorbate
 20. 11. Thestable liquid formulation of claim 10, comprising about 50 mg/ml of theVEGF antagonist, wherein no more than about 0.7% of the VEGF antagonistis degraded following storage for up to 24 months at about 5° C., asmeasured by size exclusion chromatography.
 12. The stable liquidformulation of claim 10, comprising about 75 mg/ml of the VEGFantagonist, wherein no more than about 1.5% of the VEGF antagonist isdegraded following storage for up to 15 months at about 5° C., asmeasured by size exclusion chromatography.
 13. The stable liquidformulation of claim 10, comprising about 100 mg/ml of the VEGFantagonist, wherein no more than about 1.9% of the VEGF antagonist isdegraded following storage for up to 15 months at about 5° C., asmeasured by size exclusion chromatography.
 14. The stable liquidformulation of claim 9, comprising about 3% PEG
 3350. 15. The stableliquid formulation of claim 14, comprising about 50 mg/ml of the VEGFantagonist, wherein no more than about 1.3% of the VEGF antagonist isdegraded following storage for up to 24 months at about 5° C., asmeasured by size exclusion chromatography.
 16. The stable liquidformulation of claim 14, comprising about 75 mg/ml of the VEGFantagonist, wherein no more than about 2.0% of the VEGF antagonist isdegraded following storage for up to 15 months at about 5° C., asmeasured by size exclusion chromatography.
 17. The stable liquidformulation of claim 14, comprising about 100 mg/ml of the VEGFantagonist, wherein no more than about 2.6% of the fusion protein isdegraded following storage for up to 15 months at about 5° C., asmeasured by size exclusion chromatography.
 18. A non-liquid formulationof a vascular endothelial growth factor (VEGF) antagonist made accordingto the steps of: (a) combining in a liquid solution the followingexcipients: (i) about 50 mg/ml of a VEGF antagonist of SEQ ID NO:4; (ii)about 10 mM histidine, pH about 6.3-6.5; (iii) about 1.5% polyethyleneglycol (PEG) 3350; (iv) about 5% sucrose; and (v) about 1.5% glycine;and (b) lyophilizing the combination of step (a), wherein, followingstorage of the lyophilized combination obtained after step (b) at 5° C.for about 6 months, at least about 98% of the VEGF antagonist is presentin native conformation as measured by size exclusion chromatography uponreconstitution.
 19. The non-liquid formulation of claim 18, wherein,following storage of the lyophilized combination obtained after step (b)at 5° C. for about 3 months, the VEGF antagonist comprises at leastabout 65% of maximum biological activity.
 20. The non-liquid formulationof claim 19, wherein the biological activity is the ability of the VEGFantagonist to inhibit the biological effects of human VEGF on a mouseBaf/3 VEGFR1/EpoR cell line reported as IC₅₀.
 21. The non-liquidformulation of claim 18, wherein, following storage of the lyophilizedcombination obtained after step (b) at 5° C. for about 3 months, theVEGF antagonist binds VEGF at a level of at least about 94% of maximumbinding.
 22. The non-liquid formulation of claim 21, wherein the bindingof the VEGF antagonist to VEGF is expressed as IC₅₀ as determined byELISA.
 23. A method of producing a liquid formulation of a VEGF-specificfusion protein antagonist, comprising reconstituting the lyophilizedformulation claim 18 with liquid, wherein a liquid formulation isgenerated.
 24. The method of claim 23, wherein the liquid is sterilewater or bacteriostatic water.